The cytokine Granulocyte Colony Stimulating Factor (G-CSF) treatment significantly improves the quality of life among patients with severe chronic neutropenia [Jones et al. JAMA 270: 1132-1133 (1993)]. The G-CSF is a potent endogenous trigger for the release of neutrophils from bone marrow stores and for their activation for enhanced antimicrobial activity. G-CSF has been widely evaluated in various pre-clinical models of acute illness, with generally promising results [Marshall J. C. Shock 24: 120-9 (2005)]. Due to its proven efficacy during chemotherapy cycles, the G-CSF is an important biopharmaceutical drug used in oncology. G-CSF has been cloned and expressed in various types of cells, e.g. microbial cells [Souza L. M. Science 232: 61-65 (1986); Hu Z. Y. et al. Zhongguo Shenghua Yaowu Zazhi (1999), 20: 55-57], yeast cells [Lasnik M. A. et al. Biotechnol. Bioeng. 81: 768-774 (2003); Lee S. M. et al. Korean patent KR 160934 B1 19981116], rice cells [Hong et al. Protein Expr Purif. Epub ahead of print (2005)], feline cells [Yamamoto et al. Gene 274: 263-269 (2001)], Chinese Hamster Ovary cells [Monaco L. et al. Gene. 180:145-150 (1996)], insect cells [Shinkai et al. Protein Expr Purif. 10: 379-385 (1997)], and even in transgenic goat [Ko J. H. et al. Transgenic Res. 9: 215-22 (2000)]. For pharmaceutical use the G-CSF is produced primarily in Escherichia coli [Jevsevar S. et al. Biotechnol. Prog. 21: 632-639 (2005)], where it is produced as inclusion bodies, which are insoluble aggregates of the recombinant protein in non-native conformation [Baneyx F. & Mujacic M. Nature Biotechnol. 22: 1399-1408 (2004)], that generally do not have biological activity [Bernardez C. E. Curr. Opin. Biotechnol. 9: 157-163 (1998)]. The technologies of its secretory production [Jeong K. J. & Lee S. Y. Protein Expr Purif 23: 311-318 (2001); Lee S. Y. et al. Methods Mol. Biol. 308: 31-42 (2005)], have also been reported. Secretory expression generally results into the release of properly folded form of G-CSF into the periplasmic space or extra-cellular medium, but the yields are much lesser than those obtained with inclusion bodies. It is therefore commercially beneficial to express G-CSF in E. coli as inclusion bodies. Properly folded, biologically active G-CSF protein is easily obtained from inclusion bodies in a commercially viable manner, using denaturation and renaturation processes applied subsequent to the isolation and solubilization of inclusion bodies [Rudolph R, In Protein Engineering: Principles and Practice; Cleland, J. L., Craik, S. C., Eds.; Wiley-Liss, Inc.: New York, 1996; pp 283-298; Rathore A. S. et al. J Pharm Biomed Anal. 32:1199-1211 (2003)].
One of the most efficient methods of recombinant protein production in E. coli is fed-batch, which can be carried out, in cyclic and non-cyclic modes. The non-cyclic processes are less complex and therefore more suitable for industrial production. In fact, prior art describes one of the highest GCSF yields from a non-cyclic fed-batch process which is in the range of 4.2-4.4 g/L [Yim S C et al. Bioprocess and Biosystems Engineering (2001), 24, 249-254]. Carrying out fed-batch fermentation in cyclic mode in order to obtain higher cumulative yield results in high plasmid instability [Choi S.-J. et al. J. Microbiol. Biotechnol. 10: 321-326 (2000)], thereby limiting the robustness of the process.
In general, in order to have high expression of the product it is imperative to keep the product gene-containing extra-chromosomal plasmid inside the cell in its proper form. This is generally achieved by maintaining selection pressure on the recombinant microorganism by adding a suitable antibiotic to the culture broth. Increase in expression level of G-CSF by adding antibiotic (Ampicillin) every 1-2 h during fermentation to decrease the ‘segregational nonstability’ of recombinant strain has been reported (Krivopalova G. N. et al. Russian Patent RU 2158303 C2 20001027). The regulatory requirement of the evidence of antibiotic clearance from the final product necessitates the limit of its use. Higher usage of antibiotics may also have a higher potential of having an undesirable environmental impact. But the decrease in antibiotic selection pressure often results in decreased plasmid stability and expression levels, which compromises the robustness of process. Therefore, it is a technical challenge to limit the use of antibiotic while increasing the plasmid stability and expression level of the product, especially during production phase. Further, low plasmid stability during production phase can also be due to metabolic stress [Saraswat V. et al. FEMS Microbiol. Lett. 179: 367-373 (1999)], and might lead to low expression levels [Cheng C. et al Biotechnol. Bioeng. 56: 23-31 (1997)], typically in high volume cultures.
Besides maintaining high antibiotic selection pressure, plasmid stability can be improved at the level of vector construction [Schweder T. et al. Appl Microbiol Biotechnol. 38:91-93 (1992); Pan S. H. and Malcom B. A. Biotechniques. 29:1234-1238 (2000)]. In process it can be improved by adjusting the culture conditions, such as avoiding nutrient starvation [Smith & Bidochka Can. J. Microhiol. 44: 351-355 (1998)]. While carrying out large-scale substrate-limiting fed-batch processes, nutrient limitation/starvation is imminent, and adding antibiotic either too frequently or in large amounts are also impractical and expensive solutions to maintain high product yield and high plasmid stability in a process of low complexity. Therefore, there is a clear need to develop an alternate process for preparing G-CSF in high volumetric yields by maintaining high plasmid stability using a simple and robust process.